KR101726207B1 - --glucosidase LT19-2 [KCTC18497P]Novel Bifidobacterium animalis LT19-2 strain[KCTC18497P] having excellent -glucosidase activity - Google Patents

Abstract

The present invention relates to a novel Bifidobacterium animalis LT19-2 strain [KCTC18497P] having an excellent -glucosidase activity and isolated from a Korean infant. Compared to conventional developments, the strain of the present invention has a high biological activity. In particular, the strain is isolated from intestinal secretion of a Korean, thereby having good intestinal survival rates and specific organism activity effects. Thus, it is expected that the strain can be widely used in developing a lactobacillus using processed food and a drug. The strain has an excellent -glucosidase activity of biological activities.

Description

Glucosidase activity [having excellent β-glucosidase activity] Bifidobacterium animalis LT19-2 new strain [KCTC18497P] {Novel Bifidobacterium animalis LT19-2 strain [having excellent β-glucosidase activity]

The present invention relates to a method for producing Bifidobacterium ( Bifidobacterium tuberculosis) having excellent β-glucosidase activity isolated from feces of a Korean infant The present invention relates to a novel strain [KCTC18497P], which has a specific biological activity effect different from those developed so far, especially by isolating it from the secretions of Koreans, It is expected to be widely used in the development of processed foods and pharmaceuticals, and is characterized by excellent activity of β-glucosidase activity in bioactivity and excellent acid resistance and bile resistance.

More than 80% of Koreans are known to be infected with Helicobacter pylori in the intestine, which causes inflammation on the stomach and often leads to stomach cancer. WHO also defines Helicobacter pylori as the first grade carcinogen in stomach cancer. Korea Yakult Co., Ltd., a Korean dairy company, has been developing L. acidophilus HY2177, which is an excellent urease inhibiting urease in lactic acid bacteria inhibiting Helicobacter pylori, and L We developed "Will", an excellent anti - gastric ulcer and gastritis treatment, which inhibits inflammatory cytokines by using casei HY2743. Since 2001, it has achieved sales of more than 250 billion won and has succeeded in commercialization. And the development and addition of egg yolk with the HP antibody to develop the technology to double the effect.

In addition, the amount of milk that is supplied as a main nutrient source for dairy products is higher than 1:10 of the ratio of Omega 3 to 6, which is a big difference from that of natural milk. Such milk causes obesity, It is known to be bad. As a way to improve this, Korean Yakult, which has developed a method to increase the omega 3 content in concentrated feed while reducing the rich feed intake during livestock raising and increasing the hay feed instead, has developed functional milk with a ratio of omega 3 and 6 of 1: 4 And commercialized it.

Recently, the researchers at Seoul National University have pointed out that modern people are faced with chronic metabolic diseases in which the body weight is increased due to excess of carbohydrate and energy, and the blood triglyceride, glucose and blood pressure are increased. Thus, B.lactis HY810813, Lactobacillus curvatus HY7601, Lactobacillus plantarum KY1032, and the results showed that the fat cell size was decreased, thus demonstrating the possibility of developing obese functional foods.

On the other hand, beta-glucosidase, which is known as one of the cellulases, is an enzyme which decomposes low-molecular weight water-soluble cellulose to produce glucose. It acts to liberate D-glucose units from cellobiose, cellooligosaccharide and other glucosides . That is, since beta-glucosidase plays an important role in catalyzing the final step of the process of releasing glucose from the inhibitory cellobiose, its activity and efficacy is reduced by the conversion of enzymatic lignocellulosic biomass Overall involvement, and consequently can reduce the cost of enzyme treatment. Therefore, beta-glucosidase activity has been evaluated to be closely related to health function.

Particularly, studies on increasing the absorption rate of ginsenoside of red ginseng using lactic acid bacteria have been actively carried out. When a biosynthesis using a strain having excellent activity of beta-glucosidase is used, This is because the absorption rate of the side body can be increased more efficiently. Studies on the fermentation of legumes using lactic acid bacteria have also been actively conducted.

Korean Patent Publication No. 10-0852984 discloses a novel Bacillus sp. CS90 (Bacillus sp.) Having excellent antibacterial activity and beta-glucosidase activity against acid-fast bacilli, various food poisoning microorganisms, (KACC91315P) is fermented by inoculating soybeans into soybeans. The Bacillus strain is highly likely to survive in the human body, and can be used to prevent food poisoning. In addition, By producing a large amount of polyphenol in chungkukjang produced by glucosidase activity, it is possible to produce chongkukjang having high antioxidant ability. When such a characteristic functioned as a good probiotic agent, an appropriate amount of bamboo salt and green tea powder were added to the prepared chungkukjang powder to improve the functionality and flavor.

Korean Patent Publication No. 10-1412268 discloses a beta-glucosidase mutated to increase activity and a method for producing bioethanol using the same, wherein a polynucleotide encoding a mutated beta-glucosidase to increase activity, Glucosidase expressed from a polynucleotide, an expression vector comprising the polynucleotide, a transformant transformed with the expression vector, and a method for producing the mutated bete-glucosidase using the transformant And a method for producing bioethanol using the transformant.

Thus far, research on the development of highly functional food materials through bioconversion has been actively carried out in Korea, but it is difficult to apply it to various functions, and the developed biodegradation yield of lactic acid bacteria is low, It has become a stumbling block to commercialization based on new lactic acid bacteria.

1. Published Japanese Patent Application No. 10-2013-0063525 2. Patent Publication No. 10-1621425 3. Patent Publication No. 10-0852984 4. Patent Publication No. 10-1412268

1. Isolation of strains with fibrinolytic activity and β-glucosidase activity from soybean paste and optimization of enzyme production medium (Korean Journal of Food Science and Nutrition, 33 (2), pp.439-442, 2004) 2. Isolation and Classification of Strains of β-glucosidase Producing Hosiery Roseivivax roseus (Korean Journal of Microbiology, 51 (2), pp.141-147, 2015)

Concentration and aquatic products are expected to have higher functionality than those consumed wildly in the wild, because they have different physiological activities as the numbers and positions of phytochemicals such as hydroxy, methoxy and double bonds are different . However, since most of the lactic acid bacteria used for the biotransformation are derived from Europe or most of them are not derived from human body such as kimchi, there is a problem of viability when they are used as actual food materials. Therefore, The purpose of this study is to select functional strains from the feces of Korean infants and select those with a high bioconversion effect.

In addition, the present invention aims to isolate a strain having high β-glucosidase activity because the fibrinous cellulose which we ingest is composed of b-linkage of glucose and β-glucosidase as a degradation enzyme thereof.

It is another object of the present invention to provide a strain excellent in acid resistance.

It is another object of the present invention to provide a bacterium which is excellent in bile resistance and is capable of surviving in the intestines at the time of ingestion.

The present invention provides a new strain of Bifidobacterium anilithus strain LT19-2 (KCTC18497P) isolated from feces of a Korean infant which is characterized by excellent beta-glucosidase activity in order to achieve the above object.

The processed food may be fermented milk, yogurt, red ginseng fermented food, soybean fermented food, cheese, milk, soy milk, tofu, beverage, milk powder, baby food, reproductive, And a supplementary food.

The novel strain according to the present invention is useful for the treatment of Bifidobacterium ( Bifidobacterium ), which is excellent in beta-glucosidase activity, isolated from feces of Korean infants animalis LT19-2) new strain [KCTC18497P].

BP betting according to the present invention, Te Solarium Animal less (Bifidobacterium animalis LT19-2), a new strain [KCTC18497P], can provide processed foods with excellent intestinal fiber breakdown.

BP betting according to the present invention, Te Solarium Animal less (Bifidobacterium ( KCTC18497P) can be used for the production of bioethanol and the like because the activity of beta-glucosidase is excellent and biomass decomposition can be developed more effectively.

Further, the novel strain of Bifidobacterium animalis LT19-2 according to the present invention [KCTC18497P] is excellent in β-glucosidase activity and not only for the development of related drugs, And can be used for various applications.

FIG. 1 is a graph showing the activity of the β-gulcosidase activity of a strain having no antibiotic resistance among six strains isolated from Korean infants.
FIG. 2 shows the survival rate according to the pH of the identified six strains. All of the six strains showed strong acid resistance, indicating excellent survival rate.
Fig. 3 shows the biliary properties of the six strains identified. All six strains showed strong biliary properties.
FIG. 4 shows a high intestinal adhesive ability as a result of a comparative experiment between four kinds of starting strains derived from human body and one kind of non-human derived strains.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

< Example  1.> Korean Lactic acid bacteria  Separation, identification

1. Lactic acid bacteria isolated from infant feces of Korean

In order to isolate intestinal lactobacillus, the feces of Korean infants less than 100 days were used (IRB No.1605 / 003-006). Utilizing the selected Lactobacillus genus (lactobacillus spp.) And Bifidobacterium (Bifidobacterium spp.) From the faeces of the infant badge was isolated strains.

 First, the collected fecal samples were diluted 10-fold with 0.85% NaCl, and TOS-propionate agar supplemented with De Man Rogosa, Sharpe agar (MRS broth; Difco, USA) supplemented with 0.02% sodium azide and 50 mg / L lithium mupirocin (Merck KGaA, Darmstadt, Germany) and anaerobically incubated at 37 ° C for 48 hours. The strains were selected from the different colonies formed by the culture. The selected strains were subcultured in MRS broth and BL broth and stored frozen at -80 ° C in MRS broth and BL broth containing 20% glycerol.

2. Identification of lactic acid bacteria isolated from infant feces

The biochemical characteristics of the isolated lactic acid bacteria were determined using the API 20A kit and the API-ZYM kit (BioMetrieux Co. France). In order to identify the lactic acid bacteria, As shown in Table 1 below, the isolated lactic acid bacteria were identified as Bifidobacterium animalis by 16s rRNA sequencing of Macrogen.

Biochemical reaction, enzyme, or isolate identification strain KT 11-1 KT 11-2 KT 11-3 KT 11-4 KT 11-5 LT 19-2 Biochemical reaciton Hydrolysis of: Esculin - - - - - - Gelatin - - - - - - Production of indole - - - - - - Oxidation or fermentation of: Arabinose + + + + + + Cellobiose - - - - - - Glucose + + + + + + Glycerol - - - - - - Lactose + + + + + + Maltose + + + + + + Mannitol + + + + + + Mannose - - - - - - Melezitose - - - - - - Raffinose + + + + + + Rhamnose - - - - - - Salicin + + + + + + Sorbitol - - - - - - Sucrose + + + + + + Trehalose - - - - - - Xylose + + + + + + Enzyme Catalase - - - - - - Urease - - - - - - Isolate idendification
Bifidobacterium spp . 96.80% 96.80% 96.80% 96.80% 96.80% 96.80%

As a result of examining 19 kinds of enzymatic activities such as β-Glucosidase, 6 kinds of Bifidobacterium spp. Identified above were identified as Leucine arylamidase, Acid phosphatase, α-Galactosidase, β- Galactosidase, α-glucosidase, β-glucosidase and the like. Among these, intensive research focusing on β-glucosidase closely related to human biotransformation was conducted.

Enzymes strain
KT 11-1 KT 11-2 KT 11-3 KT 11-4 KT 11-5 LT 19-2 Control - - - - - - Alkaline phosphatase - - - - - - Esterase (C4) - - - - - - Esterase lipase - - - - - - Lipase - - - - - - Leucine arylamidase - + + + + + Valine arylamidase - - - - - - Cystinearylamidase + - - - - - Trypsin - - - - - - α-Chymotrypsin - - - - - - Acid phosphatase - + + + + + Naphthol-AS-BI-phosphohydrolase - - - - - - α-Galactosidase - + + + + - β-Galactosidase + + + + + + β-Glucuronidase - - - - - - α-Glucosidase + + + + + + β-Glucosidase + + + + + + N-Acetyl-β-glucoaminidase - - - - - - α-Mannosidase - - - - - - α-Fucosidase - - - - - -

In addition, as a result of DNA sequencing on the six species of Bifidobacterium spp. Identified above, all of the Bifidobacterium animalis subsp . lactis .

No. Strain sequencing results One KT11-1 Bifidobacterium animalis subsp . lactis 2 KT11-2 Bifidobacterium animalis subsp . lactis 3 KT11-3 Bifidobacterium animalis subsp . lactis 4 KT11-4 Bifidobacterium animalis subsp . lactis 5 KT11-5 Bifidobacterium animalis subsp . lactis 6 LT19-2 Bifidobacterium animalis subsp . lactis

< Example  2. Assessment of antibiotic resistance

The microdilution method (CLSI, M07-A9) was used to determine the resistance to the antibiotics against the six isolates of Bifidobacterium spp. For this purpose, subculture was prepared by washing twice. Cells were diluted in 2 × LSM broth to 0.3 at Mcfarland 1, OD ₆₀₀ , and further diluted 300 × in 2 × LSM broth. Add 100 μl of the prepared diluent to the microplate, and add 100 μl of the 2 × antibiotic dilution diluted 2-fold. At this time, antibiotics were prepared by diluting Ampicillin, Kanamycin, Streptomycin, and Erythromycin stepwise from 1 mg / L to 128 mg / L. Minimal inhibitory concentration was determined after anaerobic incubation for 48 hours.

As a result of the microdilution method test, it can be seen that all six strains isolated as in [Table 4] are not resistant to EFSA breakpoint criteria for four antibiotics such as Ampicillin, Kanamycin, Streptomycin and Erythromycin.

Strain, MIC (mg / L) Amp Blood Str Ery KT 11-1 ≤ ≤ 1 > 128 > 128 ≤ ≤ 1 KT 11-2 ≤ ≤ 1 > 128 > 128 ≤ ≤ 1 KT 11-3 ≤ ≤ 1 > 128 > 128 ≤ ≤ 1 KT 11-4 ≤ ≤ 1 > 128 > 128 ≤ ≤ 1 KT 11-5 ≤ ≤ 1 > 128 > 128 ≤ ≤ 1 LT 19-2 ≤ ≤ 1 > 128 > 128 ≤ ≤ 1 Breakpoints for bifidobacterium animalis EFSA 2 n.r 128 One

< Example  3. > glucosidase  Active measurement

Among six strains of Bifidobacterium spp. Isolated from Korean infants, strains that do not have antibiotic resistance and those that have β-glucosidase activity using colorimetric assay against Lactobacillus casei [ATCC393] Respectively. The selected strains were subcultured in broth to perform colorimetric assay for β-glucosidase activity, and 17,000 g of the culture was centrifuged for 5 minutes, and the supernatant and cells were disrupted to use as an enzyme solution. (Sigma, USA) was added and incubated at 37 ° C for 20 min. The enzyme solution was added to 4-Nitrophenyl β-D-glucopyranoside (Sigma, USA) After stopping the reaction by adding cold 0.5 M Na ₂ CO ₃ , the reaction solution was centrifuged for 1 minute and the absorbance at 405 nm was measured to measure the β-gulcosidase activity.

As shown in [Table 5] and Fig. 1, β-glucosidase activity was found to be highest in the intracellular matrix than in other strains, and the β-glucosidase activity of LT19-2 was the highest in the intracellular matrix. Enzyme activity was confirmed. It was found that the pellet as well as the intracelluar had much better enzymatic activity than the other strains. Therefore, various processed foods can be developed using the above strains. That is, various natural products such as milk, ginseng, red ginseng, soybean, barley, etc. can be utilized for bioconversion into useful components through microbial fermentation.

Strain intracellular (Unit / ml) pellet (Unit / ml) KT11-1 43.05 + - 0.70 13.31 ± 0.22 KT11-2 41.82 ± ± 0.40 14.97 + 0.07 KT11-3 50.07 ± 0.15 14.46 ± 0.07 KT11-4 53.14 + - 1.22 14.08 + - 0.22 KT11-5 28.05 + - 0.46 14.33 + - 0.13 LT19-2 94.15 + 2.89 19.08 ± 0.22 ATCC393 - 22.28 ± 0.27

< Example  4> Selected Lactic acid bacteria Acid resistance (Acid resistance)

Six isolated strains of Bifidobacterium spp. Were subcultured in MRS broth containing 0.05% cysteine to prepare an acid resistance study. MRS broth containing 0.05% cysteine adjusted to pH 2, 2.5, 3, and 3.5 was prepared to confirm the growth of bacteria according to the pH change of the culture, and the washed cells were cultured at 2 × 10 ⁶ CFU / ml Respectively. Then, the cells were anaerobically cultured at 37 ° C for 2 hours and then removed to confirm the viable cell count. Survival rates of the strains were measured and compared by counting viable cells two hours after the inoculation.

As a result of the above experiment, as shown in Table 6 and FIG. 2, it was confirmed that Bifidobacterium enimelis 6 has a very high survival rate even in a strong acidic environment of pH 2.0 to 3.5, which is a strain isolated in the human body It is confirmed that the above-mentioned functionality can be expected when the lactic acid bacteria are indirectly ingested directly or through bioconversion.

strain (% Survival) pH 2.0 pH 2.5 pH 3.0 pH 3.5 KT11-1 99.8 100.64 99.58 100.56 KT11-2 96.57 97.77 100.76 100.56 KT11-3 79.93 98.2 100.25 101.41 KT11-4 97.68 98.44 99.13 99.98 KT11-5 99.65 97.32 99.9 100.75 LT19-2 99.41 99.43 100 98.83

< Example  5> Selected Lactic acid bacteria My bile (Bile resistance)

Six isolated strains of Bifidobacterium spp. Were prepared by subculture in MRS broth containing 0.05% cysteine. MRS containing 0.05% cysteine, including bovine bile, at 0.015%, 0.031%, 0.062%, 0.125%, 0.25%, 0.5%, 1% and 2% in order to determine the growth of bacteria by the concentration of bile acid in the culture medium agar was prepared. The washed cells were inoculated on an agar plate in an amount of 20 μl each, incubated at 37 ° C for 48 hours, and then the MIC concentration was measured.

As a result, the MIC concentration of the strain was measured at 0.25% bovine bile acid as shown in [Table 7] and FIG. 3 below. It was confirmed that bovine bile acid of less than 0.25% in all six strains was able to grow normally. In other words, it was confirmed that strains were resistant to relatively strong bile secreted from the duodenum. Therefore, It is judged to be suitable for the function.

Strain Even MIC Bovine (%) KT11-1 0.25 KT11-2 0.25 KT11-3 0.25 KT11-4 0.25 KT11-5 0.25 LT19-2 0.25

< Example  6> Selected Lactic acid bacteria  Intestines Attachment (Adhesion)

Caco-2 cell line was used by KCTC. Cell culture was performed in medium supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U / mL penicillin and 100 μg / mL streptomycin (Gibco) in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, Respectively. The cells were incubated at 37 ° C, 5% CO ₂ , and 90% humidity. Cultured cells were cultured at 2 ~ 3 days, After washing the cells with PBS, the cells adhered to the trypsin-EDTA solution were separated, and the cells were divided into 5.4 × 10 ⁵ cells / well in a 12-well plate. The cells were incubated for 20 days at a concentration of 1 × 10 ⁸ CFU / ml and cultured at 37 ° C. and 5% CO ₂ for 2 hours. After washing twice with PBS, the number of viable cells in the cells attached to the MRS medium containing 0.05% cysteine was measured. The adherence is expressed in adherence% and can be calculated by comparing the initial value with the following formula.

[Formulas]

% adhesion = [(CFU / ml _{120 min} ) / (CFU / ml 0 _min )] 100

Strain % adhesion LGG 5.58 KT11-1 10.0 KT11-2 4.082 KT11-3 10.0 LT19-2 17

As a result of the experiment, as shown in Table 8 and Fig. 4, the control group L. rhamnosus The isolates KT11-1, KT11-2, KT11-3, and LT19-2 were found to have high intestinal adhesiveness compared to LGG, and the LT19-2 strain was superior to other strains. This indicates that the strain derived from human body has a higher adaptability in the intestines than the non - human strain, and it can be used as a probiotic agent in the future.

Korea Biotechnology Research Institute KCTC18497P 20160826

Claims (3)

Bifidobacterium animalis LT19-2 new strain [KCTC18497P] isolated from feces of Korean infants characterized by excellent beta-glucosidase activity,
Processed food made from the strain of claim 1
3. The method of claim 2,
Wherein the processed food is any one of fermented milk, yogurt, fermented red ginseng, soybean fermented food, cheese, milk, soy milk, tofu, beverage, milk powder, baby food,

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